Embryo Collection and Culture

Mice were maintained on a 12 hr light/dark cycle. Preimplantation (E2.5-E4.5) embryos were collected by flushing the oviduct or uterus with M2 medium (Sigma M7167). Post-implantation (E4.75-E6.5) embryos were collected by dissecting the embryos from the decidua in ice-cold PBS containing 1% FBS (HyClone SH30396.02) or Bovine Serum Albumin (BSA, Sigma A7888). During embryo collection, dissected embryos were held in warm M2 media. For embryo culture, KSOM medium (Millipore MR-121-D) was equilibrated overnight prior to embryo collection. Where indicated, the following were included in the culture medium: 1 μM or 0.25 μM LDN-193189 in DMSO (Stemgent 04-0074-02); 1 μg/mL recombinant FGF4 in PBS with 0.1% BSA (R&D 235-F4); 1 μg/mL heparin (Sigma H3149); 100 ng/mL recombinant BMP4 in 4 mM HCl (R&D 314-BP); 1 μM PD173074 in DMSO (Selleckchem S1264); 5 μM PD0325901 in DMSO (Stemgent 04–0006); or 0.2% DMSO (New England BioLabs B0515A) as control. Embryos were cultured at 37°C in a 5% CO₂ incubator under light mineral oil (Millipore ES-005-C).